At present it is estimated that over 1,500 million cases of diarrhea occurred every year worldwide, of which, 3 million ended in death. Of the total number of cases of diarrhea, 210 million are caused by the bacterium enterotoxigenic bacterium Escherichia coli, hereinafter designated as ETEC, of these episodes 380,000 cases ended in death (World Health Organization (WHO) State of the art of new vaccines Research & Development Initiative for Vaccine Research; Geneva, April 2003). Although the diarrhea caused by this microorganism occurs in groups of all ages, the mortality is more common in children under 5 years old, particularly when this illness occurs concomitantly with malnutrition, thereby the developing countries are specially affected. This bacterium also is the principal causal agent of the so-called traveler's diarrhea.
ETEC infection is acquired orally, principally through contaminated drinks or food; the bacterium overcomes the acidic conditions of the stomach until it reaches the small intestine, where, due to its Colonization Factor Antigens known as CFA's, it adheres to the intestinal mucosa and liberates its enterotoxins which are principally two, heat-labile enterotoxin known as LT and heat-stable enterotoxin or ST which are the factors responsible for the diarrhea (Gaastra W, Svennerholm A M. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends in Microbiology. 1996; 4(11): 444-452).
More than 20 different CFA's have been described in the literature, and of these three have been identified as the more prevalent, and they are known as CFA/I, CFA/II and CFA/IV that were reported by several authors, since there was detected between 50 to 75% of ETEC strains isolated from patients suffering from diarrhea all over the world, including Mexico, the majority are grouped in the family CFA/I. (Gaastra W, Svennerholm A M. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends in Microbiology. 1996; 4(11): 444-452. López-Vidal Y, Calva J J, Trujillo A, Ponce de León A, Ramos A, Svennerholm A-M, Ruiz-Palacios G M. Enterotoxins and adhesins of enterotoxigenic Escherichia coli are they a risk factor for acute diarrhea in the community? J Infect Dis 1990; 162:442-447, and Steinsland H, Valentiner-Branth P, Gjessing H K, Aaby P, Molbak K, Sommerfelt H. Protection from natural infections with enterotoxigenic Escherichia coli: Longitudinal study. The Lancet. 2003; 362). These types of CFA's induce protective immunity against the bacterium and they are pointed out as the most important among the strategies for the development of effective vaccines. (Middlebrook J L, Dorland R B. Bacterial Toxins: Cellular mechanisms of Action. Microbiological Reviews. 1984; 48(3): 199-221. McConell M M, Hibberd M L, Penny M E, Scotland S M, Cheasty T, Rowe B. Surveys of human enterotoxigenic Escherichia coli from three different geographical areas for possible colonization factors. Epidemiol. Infect. 1991; 106: 477-484).
Due to the epidemiological importance of ETEC, many of the efforts have been directed to the prevention of the illness by obtaining an effective and safe vaccine, (Levine M M, Kaper J B, Black R E, Clements A M. New Knowledge on Pathogenesis of Bacterial Enteric Infections as Applied to Vaccine Development. Microbiological Reviews. 1983; 47(4):510-550) however, to the date these efforts have been unsuccessful and a product that satisfies these needs has not been placed in the market yet. (World Health Organization [WHO] State of the art of new vaccines Research & Development Initiative for Vaccine Research; Geneva, April 2003).
One of the vaccines that is under development and that has passed to the phase of trials in human healthy volunteers from different geographical regions, was prepared in the University of Götemburg in Sweden, and it is based on subunit B of cholera toxin combined with 5 formalin inactivated strains of ETEC, which all together express the CFA's of most epidemiological importance on a global scale. (Quadri F, Ahmed T, Ahmed F, Sack B, Sack A, Svennerholm A M. Safety and Immunogenicity of an oral, inactivated enterotoxigenic Escherichia coli plus cholera Toxin B subunit vaccine in Bangladeshi children 18-36 months of age. Vaccine 2003; 21:2394-2403).
Other strategies for the development of vaccines have focused on the use of live bacteria, as it is seen in the work realized in the Center for Development of Vaccines CVD of the University of Maryland, using Shigella as vector for the expression of colonization factors and enterotoxins from ETEC. (Barry E M, Altboum Z, Losonsky G, Levine M M. Immune responses elicited against multiple enterotoxigenic Escherichia coli fimbriae and mutant LT expressed in attenuated Shigella vaccine strains. Vaccine 2003; 21:333-340).
Recently a vaccine has been prepared with a new technology of administration; by means of a patch for transdermal immunization containing the surface component of E. coli known a CS6 and the heat-labile toxin known as LT; which has been already tested in human volunteers, where an immune response of Th1 and Th2 type characterized by IgG2a and IgG1 respectively, was determined (Wenneras C, Firdausi Q, Prodeep K B, Bladley S and Svennerholm A-M. Intestinal Immune Responses in patients Infected with Enterotoxigenic Escherichia coli and in vaccinees. Infect. Immun. 1999; 66:3311-3316. Quadri F, Ahmed T, Ahmed F, Sack B, Sack A, Svennerholm A M. Safety and Immunogenicity of an oral, inactivated enterotoxigenic Escherichia coli plus cholera Toxin B subunit vaccine in Bangladeshi children 18-36 months of age. Vaccine. 2003; 21: 2394-2403. Byrd, W., and F. J. Cassels. 2003. Mucosal immunization of BALB/c mice using enterotoxigenic Escherichia coli colonization factors CFA/I and CS6 administered with and without a mutant heat-labile enterotoxin. Vaccine 21: 1884-1893. Helander A, Wenneras C, Quadri F, Svennerholm A M. Antibody Responses in Humans against Coli Surface Antigen 6 of Enterotoxigenic Escherichia coli. Infection and Immunity. 1998; 66(9):4507-4510). However, the immune response against the colonization factors was not consistent as shown by the prevalence of the illness in different geographical regions.
There is other type of developed strategies that including vaccines formed by colonization factors encapsulated in microspheres, or by means of the expression of the subunit B of LT in plants of tobacco, potatoes, tomatoes and bananas, but these strategies are highly expensive and nevertheless the conferred protection is low.
Another option can be a vaccine prepared with the common linear epitope of immuno-dominant sequences from CFA's which offered a broader spectrum and increases the level of protection from the ETEC infection (López-Vidal Y, Epitopos continuos y comunes presentes en las fimbrias de Escherichia coli enterotoxigénica (ETEC). Gac. Med. Mex. 1997; 133 (6): 511-525. Domínguez M, et al. Colonization Factor Antigenic I Peptide as Intranasal Vaccine Approach against enterotoxigenic Escherichia coli infection in hamsters. 5th National Symposium, Basic Aspects of vaccines, 1999).
Generally we can recount that different types of vaccines have been evaluated against diarrhea caused by ETEC and although many of these vaccines have demonstrated protective effectiveness against ETEC infection, some of them show certain side effects; another important problem to be considered in the design of vaccines against ETEC, is the variability in the prevalence of the different colonization factors, which prompted us to design a vaccine that is effective and safe against the different ETEC serotypes.
Intranasal administration can be an additional alternative for a vaccine against ETEC, which would add additional advantages, providing superiority over other vaccines. In this respect a great number of studies has been performed that demonstrated that the mucosa-associated lymphoid tissue known as MALT, is a common system, that is, that the stimulation in a certain site of the mucosa also known as inductor site elicits a response at local level, remotely or in the effector site, this represented a great advantage in vaccine design, with the form of transmucosal administration being greatly facilitated (Cripps A W, Kyd J M, Foxwell A R, Vaccines and Mucosal immunization. Vaccine 2001; 19:2513-2515). The use of the intranasal route as route of administration has as principal advantages the presence of a highly vascularized greater surface, the elimination of the use of syringes with the risk that this poses, the decrease of administered doses compared to the oral route which involves a decrease of adverse effects, easy administration, application to a great number of persons in relatively short times and the induction of antibodies and cells of the immune response (Zuercher A W. Upper Respiratory Tract Immunity. Viral Immunology 2003; 3; 279-289). There are studies that demonstrate that the intranasal administration can stimulate the production of secretory IgA at intestinal level, which provides a great advantage in the development of vaccines directed to the protection against intestinal pathogens (Hong-Yin Wu, Russell M W. Induction of mucosal and systemic immune responses by intranasal immunization using recombinant cholera toxin B subunit as an adjuvant. Vaccine 1998; 16(2/3):286-292).
Vaccine strategies to improve immunity at the level of the mucosa include the use of these alternate routes together with adjuvants. (Colonization Factor Antigenic I Peptide as Intranasal Vaccine Approach against enterotoxigenic Escherichia coli infection in hamsters. Domínguez M, et al. 5th National Symposium, Basic Aspects of vaccines, 1999). Recently, nasal mucosa has been used as inductive site that has showed to increase the immune response at level of the mucosa both locally and distally such as in intestinal mucosa, respiratory mucosa and genital mucosa. Administering certain antigen to the mucosa associated to lymphoid tissue is a way of eliciting immune response in sites distal from mucosa. (Byrd, W, and F. J. Cassels. 2003. Mucosal immunization of BALB/c mice using enterotoxigenic Escherichia coli colonization factors CFA/I and CS6 administered with and without a mutant heat-labile enterotoxin. Vaccine 21:1884-1893. Van Ginkel F W, Nguyen H H, McGhee J R, Vaccines for Mucosal Immunity to Combat Emerging Infectious Diseases. Emerging Infectious Diseases. 2000; 6(2):123131.)
For the above, is still of great relevance to count with an effective vaccine against ETEC infection that can be applied globally and through a route of administration that allows a rapid, easy application and that could minimize the adverse effects that could be generated.
The present disclosure relates to a peptide vaccine that can be administered intranasally, containing a common linear protein epitope from different ETEC colonization factors that can be recognized by sera of patients infected with said bacteria.